Japan Priority Application 2001-272478, filed Sep. 7, 2001 including the specification, drawings, claims and abstract, is incorporated herein by reference in its entirety.
This invention relates to highly sensitive cells that can detect environmental pollutants as well as to the detection methods employed.
Currently, evaluation of the toxicity of environmental pollutants is accomplished by instrumental analysis or bioassays using test organisms. Instrumental analysis using, for example, HPLC, GC/MS or LC/MS, enables highly sensitive detection of environmental pollutants and these techniques have been established as a means for analyzing specific known components. Bioassays using mammalian, microbial and fish test organisms precisely reflect the effects of chemical substances on living organisms. However it is substantially impossible to test the effect of large numbers chemicals individually, without taking into account any synergistic effects multiple chemicals may have when tested together because these tests require a large number of animals. The difficulty is compounded when examining the effects of long-term exposure and effects over multiple generations. Thus, simple and economical methods of testing the effect of chemicals on a biological system are needed.
At present, some in vitro assay systems are known. Compounds are tested for mutagenicity using detection systems such as the Ames test, chromosomal aberration tests which uses cultured cells and the cytotoxicity test which measures cell growth rates. Additionally, new detection systems can detect characteristic features like estrogenic response using human breast cancer cells MCF-7 cells that express estrogen receptors. For example, vitellogenin, which usually appears in blood of female fish, is induced in male fish when they are exposed to xenoestrogen. Reporter assays are also known using cells transformed with a luciferase reporter gene.
Chemicals of social concern are not always detected by the conventional acute toxicity tests. Atmosphere, water and soil contain not only heavy metals but also various kinds of pollutants, including plastics, plasticizers, agrochemicals, plant estrogen etc., which are suspected endocrine disrupters. For example, heavy metals are known toxins and recent research reveals that some chemicals act as endocrine disrupters, but in many cases, the toxic mechanisms are unknown. It is possible our bodies are exposed to such chemicals, which are accumulated through food chains. Therefore, systems are demanded which can detect chemicals that cannot be evaluated by conventional methods or low exposure effects.
Another example is the occurrence of water blooms of phytoplankton are found in both fresh water and marine environment. Cyanobacteria blooms occur in water containing adequate levels of essential inorganic nutrients such as nitrogen and phosphorus. Some species of cyanobacteria produce toxins which are classified according to their mode of action into hepatotoxins (e.g. microcyctins, nodularins), neurotoxins (e.g. anatoxins), skin irritants and other toxins. Toxic water blooms have caused death in domestic and wild animals as well as human illness. Recently, it was reported that dialysis patients in Brazil died of acute hepatic failure due to cyanobacteria contamination of the water used. These toxins are not only present in the water, but are also accumulated in shellfish such as shrimps, prawns and lobsters. Therefore, such toxins may damage human health.
Methods of detecting microcystins are known. LC/MS provides a means of highly sensitive analysis of known individual components in a sample. It is also possible to detect microcystins using an enzyme immunoassay. Additionally, a simpler method of detecting the presence of microcystins exploits the, specific inhibitory activity of microsystins in protein phosphatase 1 and 2 activity assays in enzyme activity assays
Acute toxicities can be studied by their effect on functional disorders in animals or cultured cells. However, it requires much time and cost to obtain results with utmost confidence. Many kinds of chemicals are found in low concentrations in most environments so that it is difficult to detect these substances rapidly.
Therefore a rapid and simple assay, which enables the evaluation of effects on human and on ecosystems by endocrine disrupters and heavy metals is required. Environmental pollution has become more complicated, synergistic and long-term effects are now a serious problem. This discussion demonstrates the importance of bioassays and indicates the risk and use for the assessment.
In Chemical Abstracts, a database of chemical substances, about twenty million of substances have been registered. In Japan, about sixty thousand chemical substances are in daily use and, reportedly, not less than about ten thousand synthetic chemical substances have been or are being accumulated in the environment. Hazardous chemical substances, typically endocrine disrupters, whose toxicity can hardly be predicted by conventional toxicological methods are now known. Thus, it has become important to correctly understand the hazardous features possibly caused by those substances in order to facilitate protective measures or countermeasures.
Instrumental analysis by GC/MS or LC/MS technique can separate, identify and quantify single individual components, but are not suited as means to totally analyze or evaluate the influences of multiple components on living organisms. It is anticipated that a vast sum of time and cost will be required to individually investigate the influences of these known or unknown chemical substances occurring in trace amounts in humans or in the environment by these conventional instrumental methods. There is also a possibility that a plurality of chemical substances, each occurring in trace amounts, may produce a synergistically increased or modified influence on humans and environment. In particular, the landfill leachate of industrial wastes or the byproduct of refuse incineration contains not only known existing hazardous substances, but also unknown chemical substances. It is very difficult to investigate the hazardous features of such compounds individually, to say nothing of detecting synergistically enhanced complex contamination by a plurality of substances. Currently, however, such hazardous substances have been revealed as occurring in the environment one after another and, therefore, a method of rapidly detecting, with high sensitivity, the risk of hazardous chemical substances, including unknown chemical substances, occurring in the environment, is required.
In addition to in vivo methods using fish or mammals, are biological methods of evaluating the impact of hazardous chemical substances. Such in vitro methods, using cultured microbial or animal cells, can detect the binding of a hazardous chemical substance to a specific target protein. In some of these biological methods (bioassays), the influence of a test substance is detected by utilizing the specific response of the cell type used or of the derived tissues to the substance. In those cases, it is assumed that microorganisms, including yeasts, plants, fish and animal cells will react to the chemical substance. Furthermore, even in those systems in which human cells are used, the conventional bioassays detect the effect of trace chemical substances on cells via binding to a cell-specific receptor or receptors and as such are unable to generate complete information rapidly and with high accuracy.
In this respect, it is known that the stress response system, in particular the heat shock protein (HSP) inducing system, functions in all mammalian tissues and cells. Therefore, in bioassay systems utilizing this stress response, it is not necessary to take into consideration the basic problems regarding cell specificity. Furthermore, in the actual environment, e.g. in landfill leachate, although many pollutants are present this may only account for a few percent of total organic carbon as determined by GC/MS. Therefore, leachate possibly includes numerous unknown hazardous chemicals. In the art, there are many compounds whose toxicities are unknown, thus making it difficult to make an accurate list as well as to evaluate their hazardous effects. There has been no easily implemented, trustworthy system for accomplishing the above object using the HSP promoter. A system for quantitative detection of the common response of various cells to toxic factors and for maintenance of homeostasis in living organisms, that is to say the response to stresses, is needed. This system should prove to be a highly sensitive detection system for hazardous environmental pollutions, thus providing solutions to the above problems.
Since microcystins have a difference in toxicity between homologs, a simple evaluating system for low concentration microcystins is desired. J. Chem. Soc. Perkin Trans. 1:2311, (1984) It has reported that these biotoxins not only have acute toxicity but also serve as promoters of carcinogenesis, J. Cancer Res. Clin. Oncol 180:420(1992). However the exact mechanism of toxicity is not clearly understood. Because all natural toxic substances, including modifications of those substances, have not been identified, bioassays for detecting toxicity of an unidentified toxic substance, including analogs, or toxicity due to the presence of a plurality of natural toxic substances in low concentrations is required. This bioassay should reflect physiological conditions as much as possible and should be able to be performed rapidly. Thus, a highly sensitive total detection and evaluation assay is desired.
If the general response of various cells to toxicity or stress can be detected quantitatively in a certain system, this will be expected to be an effective method that can clear many problems for total detection of biotoxins, including microcystin.
The present invention relates to a highly sensitive system for detecting trace amounts of environmental pollutants and natural toxins. Cells were transformed with a heat shock factor binding site connected to a reporter gene to obtain a stable cell line. To assay the reporter protein, which is induced by activation of the former heat shock factor sequence, the effects of the hazardous chemicals can be quantitatively evaluated.
Thus, in a first aspect, the present invention provides a cell which is obtained by transfer of a promoter containing a heat shock factor binding DNA sequence and a transcriptional regulatory sequence necessary on an occasion of stress induction as a transcriptional regulatory factor binding site, as well as a reporter gene under the control of the above-mentioned promoter into a chromosome, said reporter gene being connected, on the downstream side thereof, to the SV40pA signal without any intron, and which is used for detecting chemicals or natural toxins which disrupt or disturb homeostasis of organisms, by measuring said reporter gene protein inducing activity.
The xe2x80x9ccell which is obtained by transfer of the gene into a chromosomexe2x80x9d refers to as a stable transformant as distinguished from a cell in a transient expression system.
As the heat shock factor binding DNA sequence, includes, for example, the heat shock factor binding sequence occurring in the human HSP promoter, the mouse HSP promoter, and the Drosophila HSP promoter.
The xe2x80x9ctranscriptional regulatory sequence necessary on an occasion of stress inductionxe2x80x9d refers to a transcriptional regulatory sequence which functions on the occasion of stress induction of a protein but not transcriptional regulatory sequence which functions independently of stress induction (specifically the AP-1 binding DNA sequence or the NF-B binding DNA sequence). Preferably, the cell of the present invention does not contain any transcriptional regulatory sequence which functions independently of stress induction. Specific examples of the transcriptional regulatory sequence necessary on the occasion of stress in-duction, include the SP1 factor binding DNA sequence and GAGA factor binding DNA sequence, which are necessary for immediate response of the promoter when the cell is placed under stress.
The promoter containing a heat shock factor binding DNA sequence and a transcriptional regulatory sequence necessary on an occasion of stress induction as a transcriptional regulatory factor binding site is not particularly restricted provided that is has the above two DNA sequences. Examples of suitable promoters include the heat shock gene promoter, HSP47 promoter and HSP100 promoter. The heat shock gene promoter is preferred. Also preferred is the HSP47 promoter containing the heat shock factor binding DNA sequence and SP1 factor binding DNA sequence.
The reporter gene is preferably one that provides for easy and rapid enzyme assay. Examples of suitable reporter genes include the xcex2-galactosidase gene and the genes coding for such proteins as luciferase, chloramphenicol acetyltransferase and alkaline phosphatase.
In the above cell, the SV40pA signal (Mol. Cell. Biol. 10:4248, (1989)) is preferably connected to the above reporter gene on the downstream side thereof. In this case, it is more preferred that no intron be introduced immediately before the SV40pA signal.
The above cell is preferably constructed from a cultured animal cell. The above cell is preferably a stable transformant derived from a Chinese hamster ovary (CHO) cell or HeLaS3 cell. The mentioned cell is preferably CHO-derived stable transformant 2F1813. (This cell strain was deposited on Aug. 17, 2001 at National Institute of Advanced Industrial Science and Technology International Patent Organism Depositary and given a domestic deposition number, FERM P-18473. It was transferred to the international deposition phase based on the Budapest Treaty and on Feb. 7, 2002, given an international deposition number, FERM BP-7876).
In a second aspect, the present invention provides a method for detecting a hazardous chemical substance and/or a natural toxic substance disrupting or disturbing homeostasis in living organisms, which comprises culturing the above cell on a test sample containing medium and then measuring a reporter gene protein inducing activity.
This method can be applied in a method of evaluating a test sample containing an endocrine disrupter, a method of evaluating a test sample obtained from the environment and containing environmental standard substances, a method of evaluating a test sample containing a heavy metal, and a method of evaluating a test sample containing two or more different hazardous chemical substances in admixture, inclusive of an unidentified substance.
Preferably, the natural toxic substance is a toxin produced by blue-green algae. More preferably the natural toxic substances are microcystins or nodularins.
In a third aspect, the present invention provides a kit for detecting a hazardous chemical substance or a natural toxic substance disrupting or disturbing homeostasis in living organisms, which comprises the above cell.
In the above kit, there may be contained reagents necessary for detecting a hazardous chemical substance or a natural toxic substance disrupting or disturbing homeostasis in living organisms, for example, a medium, a solvent in which a test sample may be dissolved and a substrate which can measure a reporter gene protein inducing activity, and so forth.